Cloning and expression of the porcine attaching and effacing-associated (paa) gene of enteropathogenic Escherichia coli.

Porcine enteropathogenic Escherichia coli (PEPEC) produce an outer membrane protein (intimin) called Paa (porcine attaching and effacing-associated), which is involved in the pathogenesis of E. coli in piglets with diarrhea. The paa gene of a PEPEC strain isolated in Paraná, Brazil, was amplified by polymerase chain reaction, sequenced, and cloned into the pTrcHisTOPO2 vector. The deduced amino acid sequence encoded by the paa gene of PEPEC from Paraná, Brazil, showed 99% homology to the sequences from other PEPEC strains. In this study, the overexpression of recombinant Paa (rPaa) using alternative induction strategies was attempted. The auto-induction protocol showed excellent results for rPaa protein production with 0.4% (w/v) lactose. The rPaa protein is insoluble and was purified with Triton X-100 wash as a total antigen. This method produced a relatively high yield of rPaa. rPaa was recognized by serum from pigs immunized with the PEPEC strain. These results suggest that rPaa could be included in the development of a vaccine against swine colibacillosis.

Paracoccidioides brasiliensis and P. lutzii antigens elicit different serum IgG responses in chronic paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (S1, PS2, and PS3) and by the new species, P. lutzii. Considering that genetic differences in the Paracoccidioides genus could elicit distinct immune responses by the host, current research investigated serum IgG levels to antigens from P. brasiliensis B339 (S1), P. brasiliensis LDR3 (PS2), and atypical strain LDR2 (P. lutzii), in patients with chronic PCM from the northern and west regions of Paraná, Brazil (n = 35). Cell-free antigen (CFA) and high molecular mass fraction (hMM) were produced from each strain. Samples were analyzed by ELISA and immunodiffusion (ID). ELISA positivity using CFA: B339-100 %, LDR3-83 %, and LDR2-74 %. Response to CFA from B339 was more intense (p < 0.05), while there was no difference between LDR3-LDR2. IgG anti-hMM was higher for antigens from B339 or LDR3, when compared with LDR2 (p < 0.05). There was a positive correlation for each strain between CFA-hMM and for hMM between B339-LDR3 and LDR3-LDR2. ID positivity with CFA: B339-63 %, LDR3-66 %, and LDR2-60 %. We conclude that the intensity of reaction of the patients' sera varies with the strain used; hMM influences tests that use CFA, independently of strain; using ID, positive rates were very similar, but there was a large number of false negative results; ELISA tests using antigens from P. brasiliensis S1 were able to detect a larger number of patients than PS2 and P. lutzii (which had a considerable number of false negative results), and therefore, its use may be more appropriate in this region of Brazil.

Soluble components of Histoplasma capsulatum var. capsulatum have hemagglutinin activity and induce syngeneic hemophagocytosis in vitro.

Histoplasma capsulatum var. capsulatum is a thermally dimorphic fungus that causes histoplasmosis. Fungal hemagglutination activity and cases of reactive hemophagocytic syndrome (RHS) have been reported in the disseminated form of disease. In the present study, soluble components of H. capsulatum var. capsulatum have been investigated for hemagglutinin activity and the capacity to induce hemophagocytosis in the mouse system. To analyze hemagglutinating activity, mouse red blood cells (RBC) (1% v/v in PBS) were incubated (37 degrees C, 1 h) with cell-free antigen (CFAg) from H. capsulatum var. capsulatum (isolate IMT/HC128) (RBC-CFAg) or previously heated CFAg (56 degrees C, 30 min) (RBC-hCFAg) or as control with PBS (RBC-PBS). Hemophagocytosis was analyzed by incubating BALB/c mouse peritoneal phagocytic cells (5 x 10(6) cells) with syngeneic RBC, sensitized or not with CFAg. In addition, mouse polyclonal antibodies were raised against syngeneic RBC-CFAg (anti-RBC-CFAg) and used to analyze CFAg chromatographic fractions (Sephadex G75/120) by immunoenzymatic assay (ELISA). Hemagglutinin activity was observed with RBC-CFAg, but not with RBC-hCFAg or RBC. Also, hemophagocytosis was observed with RBC-CFAg, but not with RBC. The anti-RBC-CFAg antibodies reacted with CFAg fractions corresponding to a molecular mass (MM) higher than 150 kDa. In conclusion, the yeast form of H. capsulatum var. capsulatum releases thermolabile soluble components with hemagglutinin activity and it has been demonstrated for the first time that soluble components of the same fungus induce syngeneic hemophagocytosis in the in vitro mouse system. Also, indirect analysis with antibodies suggests that high-MM components (>150 kDa) are responsible for the interaction with RBC.

Activation of the alternative complement pathway in canine normal serum by Paracoccidioides brasiliensis / Ativação da via alternativa do complemento em soro de cão normal por Paracoccidioides brasiliensis

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p < 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis.

O fungo dimórfico Paracoccidioides brasiliensis é o agente etiológico da paracoccidioidomicose, uma doença granulomatosa humana. Recentemente, foi relatado o primeiro caso da doença natural em cães. O sistema complemento é um importante componente efetor da imunidade humoral contra agentes infecciosos. Portanto, o objetivo deste trabalho foi avaliar a ativação da via alternativa do complemento canina pelo P. brasiliensis. Inicialmente, foi avaliada a capacidade de eritrócitos de cobaia, coelho, carneiro, galinha e suíno ativarem a via alternativa do complemento canino. Os eritrócitos de cobaia apresentaram maior capacidade de ativar a via alternative do complemento canino. A atividade hemolítica da via alternativa (AH50) foi avaliada em 27 amostras de soro de cães saldáveis e os valores médios observados foram de 87,2 AH50/ml. Não foi observada diferença significativa ao sexo e idade. A ativação da via alternativa pelo P. brasiliensis foi maior nas amostras de soro de cães adultos quando comparada aos cães filhotes e idosos (p < 0.05). Este é o primeiro relato da ativação da via alternative do complemento canino pelo fungo P. brasiliensis e sugere que pode ter um papel protetor na paracoccidioidomicose canina.

Activation of the alternative complement pathway in canine normal serum by Paracoccidioides brasiliensis.

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p ≤ 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis.

Occurrence of Antibodies to Paracoccidioides brasiliensis in dairy cattle from Mato Grosso do Sul, Brazil.

The aim of this study was to evaluate the humoral immune response in cattle immunized with Paracoccidioides brasiliensis and perform a seroepidemiological study of paracoccidioidomycosis in dairy cattle from Mato Grosso do Sul. Two animals (one steer and one heifer) were inoculated with a suspension of P. brasiliensis in Freund incomplete adjuvant. Blood samples were collected periodically to evaluate humoral immune response by immunodiffusion and ELISA, using exoantigen and gp43 as antigens, respectively. The antibody production was detected by immunodiffusion and ELISA, in both animals, 14 days after immunization. The soroepidemiologic study was carried out in 400 cattle of Mato Grosso do Sul from four municipalities: Corumbá, Dourados, Nova Andradina, and São Gabriel d'Oeste. The municipalities of Corumbá (30%) and Nova Andradina (28%) showed higher positivity than Dourados (8%) and São Gabriel d'Oeste (4%). In this study we concluded that cattle immunized with P. brasiliensis develop humoral immune response for gp43, remaining with high titers of antibodies, and that this animal species could be an epidemiologic marker of paracoccidioidomycosis.

Serological detection of antibodies against Paracoccidioides brasiliensis in dogs with leishmaniasis.

The aim of this study was to detect antibodies against Paracoccidioides brasiliensis in dogs seropositive and seronegative for leishmaniasis. Sera from 836 dogs (449 positive and 387 negative to leishmaniasis) were analysed by ELISA and the immunodiffusion test using gp43 and exoantigen, respectively. The analysis of the 836 serum samples by ELISA and the immunodiffusion test showed a positivity of 67.8 % and 7.3%, respectively, for P. brasiliensis infection. The dogs positive to leishmaniasis showed a higher reactivity to gp43 (79.9%) and exoantigen (12.7%) than the negative ones (54.0% and 1.0%, respectively). The higher reactivity to P. brasiliensis antigens may be due to cross-reactivity or a co-infection of dogs by Leishmania and P. brasiliensis. The lower correlation (0.187) observed between reactivity to gp43 and Leishmania antigen reinforces the latter hypothesis.

Reactivity of antibodies from patients with acute and chronic paracoccidioidomycosis to a high molecular mass antigen from Paracoccidioides brasiliensis.

Yeast forms of Paracoccidioides brasiliensis produce polydispersed high molecular mass (h-MM) antigens. We investigated the antibodies to an h-MM antigen from P. brasiliensis by immunoblotting and ELISA in sera from paracoccidioidomycosis (PCM) patients. IgG from the sera of chronic PCM patients was able to recognize the h-MM antigen at a higher frequency in the cell-free antigen (CFA) (8/13) than in the somatic antigen (SA) (2/13), as assessed by immunoblotting. The CFA was fractionated by Sephadex G-200 chromatography, and fraction 17 (F17) with the h-MM antigen of approximately 366 kDa was used in ELISA to analyze specific levels of IgG and IgE. Patients with the chronic form showed significantly higher levels of IgG (P<0.05) but not IgE (P>0.05) to F17 by ELISA, compared to patients with the acute form or to healthy donors. In conclusion, CFA is better than SA as a source of the P. brasiliensis h-MM antigen. This study reveals a new characteristic to differentiate between the acute and chronic forms of PCM, by demonstrating a higher level of seric IgG to h-MM antigen in chronic compared to acute PCM patients.

Antigenuria and antigenaemia in experimental murine paracoccidioidomycosis.

In this study, Swiss mice were experimentally infected with Paracoccidoides brasiliensis (Pb18) and we investigated the levels of gp43 in urine and plasma, anti-gp43 and IgG-gp43 immune complexes in plasma. These levels were correlated with the histopathological findings. Blood and urine samples were collected from mice at 7, 28, 56 and 84 days after intravenous inoculation of 10(5) yeast cells, and analysed by ELISA. The results showed increased levels of soluble gp43 in the plasma in all periods, and anti-gp43 IgG and immune complexes after day 28. High gp43 levels were detected in the urine, except for day 28, coincident with the presence of compact granulomas in lungs. All the infected mice showed fungal cells in the lungs, with initial granulomatous lesions at day 7, dissemination of lesions to other organs at day 56, and granulomas lacking the surrounding mononuclear cells infiltration, especially at days 56 and 84. Our results suggest that gp43 diffuses passively into the urine, and the determination of gp43 levels in urine samples may be a non-invasive alternative method for diagnosis and follow up of PCM. Further studies are needed to determine if the cellular immune response correlate with decreased urine gp43 levels.

Immune response in dogs experimentally infected with Paracoccidioides brasiliensis.

The aim of the present study was to evaluate the immune response of young dogs experimentally infected with Paracoccidioides brasiliensis. Six dogs were infected intravenously with P. brasiliensis and one control dog was inoculated with sterile saline. The infected animals were sacrificed in groups of two at 1, 6 and 12 months after infection. During the experimental period, the immune responses of the dogs to the fungus were followed by ELISA (IgM and IgG), by the immunodiffusion test and by the skin test with gp43. After killing the dogs, samples from several organs were submitted to histopathological analysis (H&E and Grocott stains) but the fungus was not observed in any tissue. Attempts to isolate the fungus from these tissue samples were also unsuccessful. All infected dogs, except one, reacted positively to the immunodiffusion and skin tests. All infected dogs showed a humoral immune response to the gp43 antigen detected by ELISA. The IgM and IgG response peaked by the first and second month, respectively. We conclude that young dogs appear to be resistant to the development of paracoccidioidomycosis.

Experimental paracoccidioidomycosis in dogs.

The aim of this study was to evaluate the susceptibility of dogs to develop paracoccidioidomycosis by experimental infection. Puppies were inoculated with Paracoccidioides brasiliensis by an intravenous route and two out of four died 1 week postinoculation, showing, at histopathological analysis, granulomas in the lungs, spleen and liver. P. brasiliensis was isolated from these organs. The animals that survived the infection showed a strong reaction when skin was tested with gp43, a specific antigen of P. brasiliensis. These animals were killed at 1 and 5 months after infection, and no lesions, macroscopic or microscopic, were observed in the lungs, spleen or liver; furthermore no P. brasiliensis culture was obtained from these organs. These results suggest that dogs can develop paracoccidioidomycosis and reinforces the importance of this animal as a sensitive indicator of P. brasiliensis in the environment.

Inhibition of Paracoccidioides brasiliensis by pesticides: is this a partial explanation for the difficulty in isolating this fungus from the soil?

The aim of this study was to evaluate the effect of ten pesticides on Paracoccidioides brasiliensis, and thereby to investigate the possible association between these data and the difficulty in isolating P. brasiliensis from agricultural soil. Six fungicides (Alto 100, Benlate, Captan, Dithane, Plantacol, Rovral), two herbicides (Pivot, Roundup) and two insecticides (Azodrin and Curacron) were evaluated. Five P. brasiliensis isolates from the environment (labelled group 'N,' for 'nature') and five isolates from patients (group 'P'), were grown on Sabouraud's dextrose agar, at 35 degrees C, with ten different concentrations of each pesticide. The dose of pesticide that causes 50% growth inhibition (ED50) was calculated for each isolate. All pesticides assayed inhibited P. brasiliensis in a dose-dependent manner, and great variability among ED50 values was observed in isolates from both groups. No statistically significant difference was observed between averages of ED50 from groups N and P, except with Alto 100. The inhibitory effect of pesticides on P. brasiliensis suggests that they can interfere with attempts to isolate P. brasiliensis from soil, where tonnes of pesticides are applied over large areas planted with various crops.

Canine paracoccidioidomycosis: a seroepidemiologic study.

Sera from 305 dogs were analyzed by enzyme-linked immunosorbent assay (ELISA) to determine presence of the antibody anti-gp43, which reacts to a specific antigen of Paracoccidioides brasiliensis. The dogs were divided into three groups according to their origin: urban dogs (animals with little or no contact with rural areas); suburban dogs (from the urban outskirts); and rural dogs. There was a significant difference between groups (P < 0.05). Rural dogs reacted positively in 89.5% of cases, followed by suburban (48.8%) and urban dogs (14.8%). There were no differences between male and female dogs. In an attempt to verify the feasibility of skin testing with gp43 to determine sensitization against P. brasiliensis in dogs, suburban (n = 61) and rural (n = 21) dogs were tested, showing positivity of 13.1 and 38.1%, respectively. Six dogs that had higher ELISA titers and also showed strong reactions in skin testing were killed in an attempt to isolate P. brasiliensis. The fungus was not detected by culture or histopathological analysis in these dogs, suggesting that dogs have a natural resistance or that they encounter an inoculum level that is insufficient to cause disease. These results indicate that ELISA and skin testing can be useful in the epidemiological study of paracoccidioidomycosis in dogs and that encounter with the fungus in nature is a frequent event.

Evaluation of fumonisin-aflatoxin co-occurrence in Brazilian corn hybrids by ELISA.

The natural co-occurrence of fumonisins and aflatoxins was investigated in freshly harvested corn kernels (150 samples, 62 hybrids), acquired from the Central-Southern (27 samples, 21 hybrids), Central-Western (86 samples, 51 hybrids) and Northern (37 samples, 18 hybrids) regions of the State of Paraná, Brazil using enzyme-linked immunosorbent assay (ELISA). Fumonisins were detected in 147 (98%) samples at a concentration range of 0.096 to 22.6 microg/g, while aflatoxins were detected in 17 (11.3%). All the aflatoxin-positive samples (range 38.0-460.0 ng/g) came from the Central-Western region and were co-contaminated with fumonisins. Fumonisin contamination was higher in corn from the Northern (9.85 microg/g) and Central-Western regions (5.08 microg/g), when compared with the Central-Southern region (1.14 microg/g). The overall evaluation detected 62% samples with fumonisin levels < or = 5.0 microg/g. Regional differences affected fumonisin levels in the same hybrid, regardless of Fusarium count and moisture content, suggesting interference from climatic conditions, in addition to the local predominance of toxigenic strains of the Fusarium biotype.

[The production and analysis of human anti-T-lymphocyte-receptor serum heterologous for sheep erythrocytes]. / Obtenção e análise de soro heterólogo anti-receptor de linfócitos T humano para eritrócitos de carneiro.

Anti-human T lymphocyte serum specific to the receptor for sheep erythrocytes (E) was produced by immunizing sheep with autologous erythrocytes sensitized with solubilized receptors from human lymphocytes. The anti-soluble receptors serum (anti-Rs) inhibited E-Rosette formation, identified T lymphocytes by indirect immunofluorescence, agglutinated the formalized E-soluble receptors complexes, and inhibited the E-soluble receptors interaction. This anti-Rs serum was also submitted to the affinity chromatography by protein A sepharose, and the amounts of 0.15 to 120 micrograms of purified anti-Rs IgG were added to the lymphocyte culture. The result obtained showed an induction of proliferative response of the normal human lymphocytes in the presence of 60 and 120 micrograms of anti-Rs IgG.

Molecular forms of soluble human T lymphocyte receptor for sheep erythrocytes in serum and saliva.

Using a specific serum anti-soluble T lymphocytes receptor for sheep erythrocytes (E) and SDS-PAGE, we detected radioactive bands of molecular weight 58,000 in immunoprecipitates of supernatant of heated human lymphocytes (SHL), in the supernatant of PHA stimulated lymphocyte cultures (SLC), normal human serum (NHS), and serum from cancer and uremia patients, labelled with 131I. By Sephadex G-200 chromatography, in addition to this fraction, we detected molecules of molecular weight higher than 150,000 which interact with the anti-soluble receptor serum (anti-RS), in serum from cancer and uremia patients. These molecules were detected in NHS or SHL after concentration or by prolonged exposure of SDS-PAGE with some labelled and immunoprecipitated SHL samples. The soluble receptors of molecular weights 58,000 (RS1) and more than 150,000 (RS2) were fully identical when analyzed by immunodiffusion with anti-RS serum. When submitted to immunoelectrophoresis, RS1 showed electrophoretic migration similar to that of albumin, while RS2 showed a pattern close to that of alpha 2-globulin. However, RS2 did not show antigenic relationship with IgM and was not an immune complex with IgG. Even though the presence of RS in human saliva has not yet been reported, molecules that interact with anti-RS serum have been detected in human saliva and are fully identical to molecules found in supernatant of heated human T lymphocytes and NHS. The RS molecules present in human saliva have a molecular weight and electrophoretic migration similar to those of RS1 from SLC and from human serum and have no antigenic relationship with human albumin.

Purification of soluble human T lymphocyte receptors for sheep erythrocytes.

Using a polyclonal heterologous anti-soluble E-receptor serum, we identified molecules of molecular weight circa 58,000 and 150,000. The soluble receptor molecule with molecular weight of approximately 58,000 (Rs1) was initially purified from supernatant of heated lymphocytes through chromatography on Sephadex G-200 and/or DEAE-cellulose. The soluble receptor molecule with molecular weight of approximately 150,000 (Rs2) is detected at high levels in the serum of patients with cancer and uremia. Rs1 and Rs2 present in serum from cancer patients were purified by chromatography on Sephadex G-200 and by affinity chromatography using anti-Rs1 IgG. 131I-labelled supernatant of heated lymphocytes binds to sheep erythrocytes and the elution and analysis of the molecules adsorbed showed bands of molecular weights approximately 58,000 and 150,000, confirming the receptor activity of these molecules.